This invention relates to a process of producing DNA consisting of multiple tandem repetitions of an oligonucleotide unit and a cascade nucleic acid amplification reaction producing a great number of partial and complete DNA or RNA copies thereof. The invention also relates to the application of these reactions in a method of detecting a target molecule or group at a specific site and a process for the amplification of a particular DNA sequence.
The well-known polymerase chain reaction (PCR) is a process for amplifying any specific nucleic acid sequence contained in a nucleic acid or mixture of nucleic acids. In general, the process involves a chain reaction for producing, in exponential quantities relative to the number of reaction steps involved, any specific nucleic acid sequence given (a) that the ends of the sequence are known in sufficient detail that two oligonucleotide primers can be synthesized which will hybridize to them, and (b) that a small amount of the sequence is available to initiate the chain reaction. The process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers, and extending the primers in the presence of a nucleic acid polymerase and the four necessary nucleoside triphosphates to form complementary primer extension products which act as templates for synthesizing the specific nucleic acid sequence. When the complementary strands of the nucleic acid are separated, e.g. by heating, the strands are ready to be used as templates for the synthesis of complementary strands by primer extension thus doubling the number of copies of the specific nucleic acid sequence. The steps of strand separation and extension product synthesis can be repeated as often as needed to produce the desired quantity of the specific nucleic acid sequence. This basic process is described and claimed in U.S. Pat. No. 4,683,202, and variants thereof are described and claimed in the related U.S. Pat. Nos. 4,683,195 and 4,800,159.
Becton Dickinson has described a variant of the PCR technique where the thermocycling is replaced by an enzymatic destruction of the primers, thus freeing the target sequence originally binding the primer to make it able to bind a new primer (EP 0 497 272 A1, EP 0 500 224 A2, EP 0 543 612 A2). After binding of the second primer to the target sequence, the product generated by chain elongation from the first primer is removed by strand displacement as the second primer is elongated. Like PCR, this reaction employs primers annealing to both ends of a biological DNA molecule with the purpose of amplifying the intervening biological sequence. This is unlike the present DNA cascade which relies on primers annealing along the length of a constructed tandemly repeated sequence (referred to as xe2x80x9cpolymerxe2x80x9d).
Strand displacement is also involved in other DNA techniques such as the commonly used random priming labeling of hybridization probes. However, in this approach all DNA present can serve as template for the reaction. This is unlike the present DNA cascade, which is restricted to a specific pre-selected template.
PRINS reactions can also be enhanced by strand displacement DNA synthesis after destruction of already elongated primer, as described by this inventor and patented by Boehringer Mannheim. Like the Becton Dickinson reaction, this produces multiple copies of a biological target sequence, but does not have the characteristics of the present DNA cascade reaction.
J. W. IJdo et al., Nucleic Acids Research, Vol. 19, No. 17, p. 4780 (1991), report the rapid generation of human telomere repeat sequence (TTAGGG)n, with fragment sizes up to 25 kb, using a technique related to the polymerase chain reaction (PCR). The reaction is carried out in the absence of template using primers (TTAGGG)5 (SEQ ID NO:1) and (CCCTAA)5(SEQ ID NO:2). Staggered annealing of the primers provides a single strand template for extension by Taq polymerase. The primers serve as both primer and template in the early cycles, whereas the newly formed sequences serve as primer and template in subsequent stages of the reaction resulting in a heterogeneous population of molecules consisting of repeat arrays of various lengths.
The DNA synthesized is only used as a probe for hybridization, and the approach thus serves as an alternative to other procedures for labeling of hybridization probes (like end-labeling or tailing). Unlike the approach described here, no surplus short primer is added to the resulting polymers to release a cascade reaction.
A commonly used method for randomly amplifying human DNA is called alu-PCR. This approach utilizes the fact that the human genome contains certain interspersed repeated elements called alu-repeats. These closely similar elements are on the average found once every ca. 10 kb of human genomic DNA. Though the actual distance between two neighboring alu-elements differ significantly along the genome, most of these elements are situated close enough to their neighbors to enable amplification by PCR of the intervening non-alu sequence after hybridization of primers to the alu-sequence.
British Technology Group Ltd has described a similar approach for the detection of Bovine Encephalitis viruses by PCR (WO 9304198 A1). In this case the interspersed repeat is comprised of a tandemly repeated sequence containing six base monomers, each having a sequence exhibiting a dyad symmetry. It is consequently possible to amplify the intervening sequences using only one primer (binding to both strands) rather than the two primers normally employed in other types of PCR, such as the alu-PCR. The fact that the naturally occurring repeat, which is detected by this technique, holds a dyad symmetry entity, gives it a possible chance similarity to one variant of the polymer synthesized by the reactions described here. In such cases where the intervening sequences are sufficiently short, the bovine virus DNA should thus be able to serve as the template for a DNA cascade. However, such a possibility is not recognized in the British Technology Group Patent, which only refers to PCR as the resulting amplification reaction. The chance similarities also imply that both the bovine virus test and some variants of the DNA cascade make use of primers with a dyad symmetry. However, whereas these primers in the DNA cascade are used to construct a molecule, and work on the constructed molecule, the primers in the bovine encephalitis test are only thought of as probes for the diagnostic detection of certain naturally occurring DNA molecules.
In the Japanese unexamined Patent Application, publication no. 04-262799, belonging to Toyobo Co. Ltd., Toshiya and Yutaka have described the formation from a circular DNA molecule of a polymer like the one used as starting material for the present DNA cascade. They obtain the DNA circle by circularizing a designed linear DNA molecule onto a biological DNA molecule, using the circularization as a test for the presence of the relevant biological molecule. After circularization of the test molecule, they add a third DNA molecule capable of binding to the part of the test molecule that did not hybridize with the biological molecule. This third molecule then serves as a primer for rolling circle replication of the circularized test molecule, thus forming a tandem repeat polymer derived from this. In this approach it is not envisioned that the polymer thus generated could be used as the starting material for a DNA cascade. Neither is it suggested that the circularization process could be positioned at the 3xe2x80x2-end of the biological molecule, such that this end could be used as a primer for the rolling circle replication, eliminating the need for the addition of a third DNA molecule to prime this, nor that the reaction could be inverted, such that it is the biological molecule, which is circularized.
Till now, the very successful techniques for the enzymatic amplification of DNA have been designed to amplify nucleic acid sequences of biological origin to enable studies of or with these sequences. The present invention represents a new strategy, termed a xe2x80x9cDNA cascadexe2x80x9d, which is to amplify synthetic DNA. The amplification on process may then secondarily be used as a marker in biological analyses, and to co-amplify nucleic acid sequences of biological origin.
The DNA cascade is a technique for the production of multiple partial or complete copies of a preformed template. This is obtained after the initial construction (xe2x80x9clinear multiplication reactionxe2x80x9d, phase 1) of a suitable template which consists of multiple tandem repetitions of an oligonucleotide unit, each of which can per se serve as a specific starting point for the copying process (the xe2x80x9ccascade amplification reactionxe2x80x9d, phase 2).
The template for the cascade reaction may be built from two complementary oligonucleotides with an internal repetition unit in a manner similar to that described by J.W. IJdo et al., loc. cit.
However, the template is most conveniently produced by a novel process according to the invention from one oligonucleotide comprising at least one and a halt and preferably two units of a nucleotide sequence showing dyad symmetry.
This process involves repeated denaturation and annealing events to enable the oligonucleotide to grow stepwise by primed synthesis catalyzed by a DNA polymerase in the presence of the necessary nucleoside triphosphates.
This repeated denaturation and annealing can be achieved by thermocycling as illustrated in example 1, but could also be achieved by other means. One possibility would be to incubate the oligonucleotide(s) at the melting point of their duplex form (or slightly above this temperature). This would result in a statistical equilibrium, where a fraction of the molecules at any given time could support chain elongation, and thus polymer growth. In such a setup the temperature cycling would be replaced by a temperature gradient forcing the molecules to become longer and longer to accommodate for the increasing incubation temperature. The advantage of the gradient approach is that it does not require incubations at high temperatures, especially not if the DNA sequences chosen are rich in adenine and thymine. The avoidance of high incubation temperatures may be of advantage if the polymer formation is performed while the oligonucleotides are attached to specific detection reagents like avidin or antibodies, as such molecules tolerate high temperatures poorly.
Thus, in a first aspect the present invention provides a process for producing DNA consisting of multiple tandem repetitions of an oligonucleotide unit, wherein an oligonucleotide comprising at least one and a half unit of a nucleotide sequence showing dyad symmetry is copied stepwise by means of a template- and primer-dependent DNA polymerase in the presence of the necessary nucleoside triphosphates during repeated cycles of denaturation and annealing, the chain elongation taking place each time the annealing results in a frame-shifted hybridization giving rise to duplexes with buried 3xe2x80x2 ends.
The sequence of bases in the oligonucleotide could be freely chosen according to the individual needs, but in order to be able to participate in the polymerization process, the oligonucleotide must consist of at least one and a half copy of the sequence intended to be the repeating unit of the polymer. Furthermore, it may be desirable to construct the oligonucleotide such that it consists of repeats of a sequence showing dyad symmetry, since this makes the sequence complementary to itself and eliminates the need for the inclusion of a second (complementary) oligonucleotide in the polymerization process. Thus, the shortest repeating unit showing dyad symmetry would be two complementary bases, for instance the sequence xe2x80x9cATxe2x80x9d. One and a half unit of this sequence would be xe2x80x9cATAxe2x80x9d, and the shortest oligonucleotide able to serve as a substrate for the polymerization on its own would thus be a three base oligonucleotide like xe2x80x9cATAxe2x80x9d. Any repeating dyad symmetry unit larger than two bases and anyone number of dyad symmetry units larger than one and a half could also be chosen, the only limitation being the technical limitations on the size of the oligonucleotide imposed by the process used to produce the oligonucleotide. Preferably, the starting oligonucleotide comprises at least two units of the nucleotide sequence showing dyad symmetry.
In a particular embodiment of the process for producing the template the nucleotide sequence showing dyad symmetry comprises the promoter region for an enzyme capable of template-dependent DNA or RNA synthesis without the need for a primer and the complementary repeat of said region. The presence of such a promoter region in each oligonucleotide unit of the template may be of advantage in the carrying out of the subsequent cascade phase as explained below.
In another particular embodiment of the above process any nucleotide sequence to be amplified is inserted between the copies of the nucleotide sequence showing dyad symmetry in the starting oligonucleotide. If such inserted nucleotide sequence comprises the promoter region for an enzyme capable of template-dependent DNA or RNA synthesis without the need for a primer, the same result is obtained as in the first particular embodiment above.
A nucleic acid template consisting of multiple tandem repetitions of an oligonucleotide unit can also be produced by another novel process according to the invention which involves circularization of one oligonucleotide so that it has no end and thus can act as a template for an endless copying process catalyzed by an enzyme that displaces rather than digests DNA or RNA occupying the part of the circular oligonucleotide which it is about to copy producing a large molecule being a multimer of the oligonucleotide.
Thus, in a second aspect the present invention provides a process for producing nucleic acid consisting of multiple tandem repetitions of an oligonucleotide unit, wherein a circular oligonucleotide comprising at least one copy of said unit is used as a template for an endless copying process by means of a nucleic acid polymerase, which is capable of strand displacement and is substantially without 5xe2x80x2-3xe2x80x2 exonuclease activity, in the presence of the necessary nucleoside triphosphates and, if necessary, a primer capable of binding to some portion of the oligonucleotide.
The circularization process can be of two kinds, as the reaction can be designed to circularize any of the two strands on the other. If using a synthetic sequence and a biological sequence, one could thus choose to circularize the biological sequence on the synthetic or the synthetic on the biological, all depending on the design of the experiment. Likewise, one could either circularize the strand to be circularized at the 3xe2x80x2-end of the template strand, such that this could serve also serve as primer for the polymer formation, or one could do the circularization away from the 3xe2x80x2-end of the template, such that the addition of a separate primer for the rolling circle replication would be necessary.
When a polymerase capable of template- and primer-dependent DNA or RNA synthesis is used, the copying is started from a primer binding to some portion of the circular oligonucleotide.
With a view to a subsequent cascade reaction the polymerase is preferably a template- and primer-dependent DNA polymerase, and it may be of advantage that the circular oligonucleotide comprises a DNA sequence showing dyad symmetry, and the primer has the same DNA sequence.
When a template-dependent RNA polymerase without the need for a primer is used, the copying is started from a promoter region incorporated in the circular oligonucleotide and being recognized by the polymerase.
In that case, if it is desired to carry out a subsequent cascade reaction, it is necessary to produce a DNA multimer from the resulting RNA multimer by means of a reverse transcriptase and a DNA primer.
For purposes of monitoring the linear multiplication reaction and detecting the multimer product it may be useful that the nucleoside triphosphates present are labeled. Such label can for example be an enzyme, a radioactive isotope, a fluorescent compound, a chemiluminescent compound, a bioluminescent compound, a metal chelate or a hapten detectable by a specific secondary reaction.
The cascade amplification reaction comprises a copying of the template in an enzyme catalyzed process that originates from multiple repeating units in the template, thus making it possible to produce multiple copies of any segment of the template. To obtain this it is necessary to use enzymes that displaces rather than digests DNA or RNA occupying the part of the template which it is about to copy. As the sequences of the produced copies are both identical and complementary, they are able to aggregate forming large complexes with a decreased mobility relative to the individual molecules.
Accordingly, in a second aspect the present invention provides a cascade nucleic acid amplification reaction, wherein a great number of partial and complete DNA or RNA copies of a DNA template consisting of multiple tandem repetitions of an oligonucleotide unit is produced by means of a nucleic acid polymerase, which is capable of strand displacement and is substantially without 5xe2x80x2-3xe2x80x2 exonuclease activity, by contacting the template with said nucleic acid polymerase in the preserve of the necessary nucleoside triphosphates and, if necessary, a primer capable of binding to the oligonucleotide unit, the polymerase thus synthesizing DNA or RNA originating from, ideally, each repeating oligonucleotide unit in the template.
If any part of the repeating oligonucleotide unit corresponds to the promoter of an enzyme capable of template-dependent DNA or RNA synthesis without the need for a primer as a starting point for the process, like the T3, T7 or SP6 RNA polymerase, the cascade phase can be induced by the simple addition of this enzyme and the necessary nucleoside triphosphates to the single-stranded or double-stranded template, preferably the double-stranded template.
If this is not the case, a primer capable of binding to the repeating oligonucleotide unit is needed along with a suitable enzyme that can synthesize DNA or RNA from the appropriate nucleoside triphosphates in a template- and primer-dependent reaction and has the aforementioned ability to induce strand displacement. In this case the strands of the template must first be separated so that the primer is able to hybridize to each strand. Suitable DNA polymerases of this kind are e.g. the Klenow fragment of DNA polymerase I, preparations of the Taq polymerase without exonuclease activity or the T4 DNA polymerase.
If the DNA template is produced from a circular oligonucleotide by means of a DNA polymerase starting from a primer binding to some portion of the circular oligonucleotide, the cascade reaction may be carried out simultaneously with the template formation by adding a primer binding to at least a portion of the complementary oligonucleotide units comprising the template.
In this case, as mentioned previously, it is advantageous that the starting circular oligonucleotide comprises a DNA sequence showing dyad symmetry, and the primer has the same DNA sequence, as then both the template formation and the cascade reaction therefrom will take place using the same single primer.
When the nucleic acid polymerase is a DNA polymerase, the synthesized strands displaced from the template are also DNA, and the cascade reaction proceeds further from the repeated oligonucleotide units of the newly synthesized DNA strands.
In a particular embodiment of such a cascade reaction the time of conducting the cascade reaction is adjusted to the number of repeated units in the template and, possibly, the concentration of primer in such a way that the copying of the template and the newly synthesized DNA strands does not proceed to the ends thereof, so that the displaced strands remain attached to the template, forming a large web of interconnected strands.
When the nucleic acid polymerase is a RNA polymerase, the synthesized strands displaced from the template are RNA, and the cascade reaction produces a great number of single-stranded RNA molecules which hybridize to each other forming a large immobile network.
The synthesized RNA molecules will not be copied further by the RNA polymerase, but if further copies are desired, it is possible to proceed as follows: The produced network of hybridized RNA molecules is denatured, annealed to complementary oligonucleotides suitable as primers for cDNA synthesis and copied into cDNA strands by means of a reverse transcriptase, after which the cascade reaction proceeds further from the repeated oligonucleotide units of the cDNA strands.
Also in the cascade reaction it may be useful for purposes of monitoring the reaction or detecting the product or products that the nucleoside triphosphates present are labeled. Again, such label can for example be an enzyme, a radioactive isotope, a fluorescent compound, a chemiluminescent compound, a bioluminescent compound, a metal chelate or a hapten detectable by a specific secondary reaction.
An application aspect of the present invention provides a method of detecting a target molecule or group at a specific site, wherein
a) a detector molecule that binds specifically to the target is attached to an oligonucleotide capable of taking part in a reaction to form a DNA template consisting of multiple tandem repetitions of said oligonucleotide,
b) the oligonucleotide with attached detector molecule is contacted with the target site, and oligonucleotide with attached detector molecule not bound to target is removed,
c) a reaction to form a DNA template consisting of multiple tandem repetitions of the oligonucleotide bound to the detector molecule is carried out, and
d) the target is detected by detection of the bound amplified nucleic acid.
In this method it will often be expedient that further a cascade reaction as previously described is carried out before detecting the target.
In another embodiment of this method
a) a detector molecule that binds specifically to the target is attached to a DNA template consisting of multiple tandem repetitions of an oligonucleotide unit,
b) the template with attached detector molecule is contacted with the target site, and template with attached detector molecule not bound to target is removed,
c) a cascade reaction as previously described is carried out, and
d) the target is detected by detection of the bound amplified nucleic acid.
When the method comprises a cascade reaction, the presence of a large web of nucleic acid strands may be visible or detectable on its own, but usually the nucleoside triphosphates used in the process for producing the DNA template and, possibly, in the cascade reaction are labeled, and the target is detected by detecting the label.
The label on the labeled nucleoside triphosphates can for example be an enzyme, a radioactive isotope, a fluorescent compound, a chemiluminescent compound, a bioluminescent compound, a metal chelate or a hapten such as biotin detectable by a specific secondary reaction.
If the product of the detection reaction shall appear at a certain localization, the target molecules or groups to be detected should be bound to a specific site either before or after the reactions according to this invention take place. For example, they may be fixed to a solid surface, or they may be confined within a narrow space such as an organic cell.
A practical use of this aspect of the invention is the one wherein the target is a specific antigen, and the detector molecule is an antibody to said antigen. Another is the one wherein the target is a specific carbohydrate molecule or group, and the detector molecule is a lectin binding thereto. Yet another is the one wherein the target is a specific nucleic acid sequence, and the detector molecule is a DNA or RNA probe which hybridize specifically to the target sequence.
A further application aspect of the present invention provides a process for the amplification of a particular DNA fragment, wherein a first oligonucleotide is added to both ends of one copy of said DNA sequence and a second oligonucleotide complementary to the first one is added to both ends of another copy of said DNA sequence, and the resulting DNA sequences are copied stepwise by means of a template- and primer-dependent DNA polymarase in the presence of the necessary nucleoside triphosphates during repeated cycles of denaturation and annealing, the chain elongation taking place each time the annealing results in a frame-shifted hybridization giving rise to duplexes with buried 3xe2x80x2 ends.
In another embodiment of this amplification process, a first oligonucleotide is added to the 5xe2x80x2 end and a second oligonucleotide complementary to the first one is added to the 3xe2x80x2 end of one copy of said DNA sequence and vice versa with another copy of said DNA sequence, and the resulting DNA sequences are copied stepwise by means of a template- and primer-dependent DNA polymerase in the presence of the necessary nucleoside triphosphates during repeated cycles of denaturation and annealing, the chain elongation taking place each time the annealing results in a frame-shifted hybridization giving rise to duplexes with buried 3xe2x80x2 ends.
In yet another embodiment of this amplification process, at least one unit of an oligonucleotide showing dyad symmetry is added to both ends of said DNA sequence, and the resulting DNA sequence is copied stepwise by means of a template- and primer-dependent DNA polymerase in the presence of the necessary nucleoside triphosphates during repeated cycles of denaturation and annealing, the chain elongation taking place each time the annealing results in a frame-shifted hybridization giving rise to duplexes with buried 3xe2x80x2 ends.
In each of the above three embodiments it may be expedient that the oligonucleotide units added to the ends of the particular DNA sequence are designed to contain restriction enzyme recognition sites bordering said DNA sequence.
In still another embodiment of the amplification process the particular DNA sequence to be amplified is either circularized or inserted into a circular oligonucleotide, and the resulting circular DNA is used as a template for an endless copying process by means of a nucleic acid polymerase capable of strand displacement and substantially without 5xe2x80x2-3xe2x80x2 exonuclease activity in the presence of the necessary nucleoside triphosphates and, if necessary, a primer capable of binding to some portion of the oligonucleotide.
In this embodiment it may be expedient that the particular DNA sequence is inserted in a site of the circular oligonucleotide producing restriction enzyme recognition sites bordering said DNA sequence.
Each of the above described embodiments of the amplification process will produce by far the largest amplification when the process further comprises a cascade reaction as previously described.
Also in this amplification aspect of the invention it may be useful for monitoring or detection purposes that the nucleoside triphosphates used in the process are labeled.